Lipofectamine rnaimax tranfection reagent

Transfection of siRNAs was performed as previously described (32 (link)). Briefly, the cells were transfected with siRNAs targeting GRP78 (si78) or control scrambled (siCtrl) using Lipofectamine RNAiMAX Tranfection Reagent (ThermoFisher Scientific, Waltham, MA). Cell lysates were harvested 48 hr after the transfection for Western blot analysis. Custom siRNAs were purchased from GE Healthcare Dharmacon, Inc. (Chicago, IL). The sequences of the siRNAs are as follow: siCtrl: 5’-GAGAUCGUAUAGCAACGGU-3’; si78 (CDS): 5’-GGAGCGCAUUGAUACUAGA-3’; si78 (3’UTR): 5’-CUUAAGUCUCGAAUGUAAU-3’.

Pre‐designed Silencer® Select siRNAs against CD13 (s683), LAP3 (s27311) and Silencer® Select Negative Control No. 1 (ThermoFischer Scientific), as well as siRNA against DPP7 (Santa Cruz Biotechnology) were used at a concentration of 10 nmol L^‐1. For delivery of siRNA, Lipofectamine® RNAiMAX tranfection reagent (ThermoFischer Scientific) was used according to the manufacturer´s protocol. 48 h post‐transfection cells were incubated with the indicated drug concentrations for 30 min, and then cell viability was monitored using an IncuCyte ZOOM (Sartorius) or using a colorimetric assay with PrestoBlue^TM cell viability reagent (Invitrogen) 48 h post‐incubation. Successful knockdown of target genes at the time point of drug application was confirmed by qRT‐PCR and Western blot.